The long term objective of this project is an understanding of the way in which tRNA is biosynthesized and the contribution of specific structural features in tRNA to tRNA function. This is to be accomplished by the modification of tRNA's by chemical and enzymatic means to provide species that can help to define tRNA's processing and function at molecular level. For the next three-year period, tRNA's will be modified at the 3'-terminus to permit studies of the spatial requirements of the aminoacyl moieties of the A-site and P-site tRNA's during the peptidyltransferase reaction. Other modifications will be carried out by altering the size of the anticodon loop, to permit an assessment of the factors controlling codon-anticodon interaction, and to provide substrates for biosynthetic studies. A new approach to RNA sequencing is also proposed.